Acute effects of strength exercises and effects of regular strength training on cell free DNA concentrations in blood plasma.

Creatine kinase (CK) is a marker for muscle cell damage with limited potential as marker for training load in strength training. Recent exercise studies identified cell free DNA (cfDNA) as a marker for aseptic inflammation and cell damage. Here we overserved in a pilot study the acute effects during strength exercise and chronic effects of regular strength training on cfDNA concentrations over a period of four weeks in three training groups applying conservation training (CT) at 60% of the 1 repetition maximum, high intensity-low repetition training (HT) at 90% of the 1 repetition maximum and differential training (DT) at 60% of the 1 repetition maximum. EDTA-plasma samples were collected before every training session, and on the first and last training day repeatedly after every set of exercises. CfDNA increased significantly by 1.62-fold (mean (±SD) before first exercise: 8.31 (2.84) ng/ml, after last exercise 13.48 (4.12) ng/ml) across all groups within a single training session (p<0.001). The increase was 1.77-fold higher (mean (±SD) before first exercise: 12.23 (6.29) ng/ml, after last exercise 17.73 (11.24) ng/ml) in HT compared to CT (mean (±SD) before first exercise: 6.79 (1.28) ng/ml, after last exercise 10.05 (2.89) ng/ml) (p = 0.01). DNA size analysis suggested predominant release of short, mononucleosomal DNA-fragments in the acute exercise setting, while we detected an increase of mostly longer, polynucleosomal cfDNA-fragments at rest before the training session only at day two with a subsequent return to baseline (p<0.001). In contrast, training procedures did not cause any alterations in CK. Our results suggest that during strength exercise short-fragmented cfDNA is released, reflecting a fast, aseptic inflammatory response, while elevation of longer fragments at baseline on day two seemed to reflect mild cellular damage due to a novel training regime. We critically discuss the implications of our findings for future evaluations of cfDNA as a marker for training load in strength training.

Internet-based perioperative exercise program in patients with Barrett's carcinoma scheduled for esophagectomy [iPEP - study] a prospective randomized-controlled trial.

BACKGROUND: Patients undergoing surgery for esophageal cancer have a high risk for postoperative deterioration of lung function and pulmonary complications. This is partly due to one-lung ventilation during thoracotomy. This often accounts for prolonged stay on intensive care units, delayed postoperative reconvalescence and reduced quality of life. Socioeconomic disadvantages can result from these problems. Physical preconditioning has become a crucial leverage to optimize fitness and lung function in patients scheduled for esophagectomy, in particular during the time period of neoadjuvant therapy. METHODS/STUDY DESIGN: We designed a prospective multicenter randomized-controlled trial. The objective is to evaluate the impact of an internet-based exercise program on postoperative respiratory parameters and pneumonia rates in patients with Barrett's carcinoma scheduled for esophagectomy. Patients are randomly assigned to either execute internet-based perioperative exercise program (iPEP), including daily endurance, resistance and ventilation training or treatment as usual (TAU). During neoadjuvant therapy and recovery, patients in the intervention group receive an individually designed intensive exercise program based on functional measurements at baseline. Personal feedback of the supervisor with customized training programs is provided in weekly intervals. DISCUSSION: This study will evaluate if an intensive individually adapted training program via online supervision during neoadjuvant therapy will improve cardiorespiratory fitness and reduce pulmonary complications following esophagectomy for Barrett's cancer. TRIAL REGISTRATION: NCT02478996 , registered 26 May 2015.

Increases in Circulating Cell-Free DNA During Aerobic Running Depend on Intensity and Duration.

PURPOSE: Increases in concentrations of circulating cell-free DNA (cfDNA) have recently been demonstrated to occur in a variety of exhausting and vigorous exercise settings. Here, the authors assessed the association of cfDNA with exercise duration and intensity in a controlled test-retest setting of a regenerative up-to-moderate-level aerobic run. METHODS: In a pretest, the lactate threshold (LT) was determined in 13 participants (range 10.8-13.4 km/h) by using a step-wise incremental running test. The speed of the 2 endurance runs was set to 9.6 km/h for 40 min; for the participants with an LT below the median (12.8 km/h; G1), this was a moderate aerobic run, and for those with an LT above the median, this was a regenerative run (G2). Capillary cfDNA, lactate, and rating of perceived exertion (RPE) were assessed before, every 10 min during, and after the runs. RESULTS: During the last 30 min of the 2 runs, lactate did not increase, whereas cfDNA increased steadily (3.46-fold for G1 and 2.05-fold for G2). Intraclass correlation for cfDNA was high (r = .81, P < .0001) for all runners but higher for male participants (r = .92, P < .0001). The correlations of cfDNA and lactate with RPEs were r = .58 (P < .0001) and r = .32 (P < .05), respectively. CONCLUSIONS: Both duration and level of intensity were significantly associated with accumulation of cfDNA. The correlation with RPE and the high test-retest reliability suggest that cfDNA might be applicable as a marker to monitor individual training load for aerobic and intermittent exercises. Future randomized, controlled, longitudinal training studies will have to reveal the full potential of cfDNA as an exercise-physiology marker.

Exploring the Potential of Cell-Free-DNA Measurements After an Exhaustive Cycle-Ergometer Test as a Marker for Performance-Related Parameters.

PURPOSE: Intensive exercise is known to be accompanied by a rapid release of cell-free DNA (cfDNA). The physiological significance of cfDNA release for performance diagnostics has not been studied. The authors analyzed the release of cfDNA during bicycle exercise and its correlation with physiological parameters. METHODS: Eleven male athletes performed an incremental cycling test. Venous blood was collected before and immediately after exercise and after 90 min of recovery. Since the amount of cfDNA is influenced by preanalytical parameters like DNA extraction and quantification method, the authors applied different measurement approaches based on quantitative real-time polymerase chain reaction. They compared a direct measurement procedure not requiring cfDNA extraction for a short (L1PA290) and a long fragment (L1PA2222) and a procedure for extracted cfDNA for a short (LTR570) and long fragment (LTR5323) with primers targeting the repetitive sequences L1PA2 and LTR5 in both assays, respectively. RESULTS: With the exception of LTR5323, the procedures revealed significant increases of cfDNA postexercise, whereas the direct approach showed lower interindividual variance in cfDNA values. When linking cfDNA levels to parameters of exercise performance the authors observed that, especially, the measurement based on L1PA2222 correlated significantly with exercise markers. These correlations were similar to the relationship of the performance markers among themselves. CONCLUSIONS: cfDNA is a possible physiological marker to assess and predict exercise performance in athletes. In addition, the results indicate that using cfDNA as a marker in exercise physiology requires careful selection of a suitable measurement technique, whether it is eluted DNA or directly quantified.

Analysis of Injury Incidences in Male Professional Adult and Elite Youth Soccer Players: A Systematic Review.

CONTEXT: The incidence of injury for elite youth and professional adult soccer players is an important concern, but the risk factors for these groups are different. OBJECTIVE: To summarize and compare the injury incidences and injury characteristics of male professional adult and elite youth soccer players. DATA SOURCES: We searched MEDLINE and Web of Science using the search terms elite, international, European, soccer, football, injury, injuries, epidemiology, incidence, prevalence, not female, not American football, and not rugby. We also used the search terms professional for studies on professional adult soccer players and high-level, soccer academy, youth, adolescent, and young for studies on elite youth soccer players. STUDY SELECTION: Eligible studies were published in English, had a prospective cohort design, and had a minimum study period of 6 months. To ensure that injury data were assessed in relationship to the athlete's individual exposure, we included only studies that reported on injuries and documented exposure volume. DATA EXTRACTION: Two independent reviewers applied the selection criteria and assessed the quality of the studies. DATA SYNTHESIS: A total of 676 studies were retrieved from the literature search. Eighteen articles met the inclusion criteria: 6 for elite youth and 12 for professional adult soccer players. CONCLUSIONS: Injury rates were higher for matches than for training for both youth and adult players. Youth players had a higher incidence of training injuries than professionals. Efforts must be made to reduce the overall injury rate in matches. Therefore, preventive interventions, such as adequately enforcing rules and focusing on fair play, must be analyzed and developed to reduce match-related injury incidences. Reducing training injuries should be a particular focus for youth soccer players.

Physical exercise induces rapid release of small extracellular vesicles into the circulation.

Cells secrete extracellular vesicles (EVs) by default and in response to diverse stimuli for the purpose of cell communication and tissue homeostasis. EVs are present in all body fluids including peripheral blood, and their appearance correlates with specific physiological and pathological conditions. Here, we show that physical activity is associated with the release of nano-sized EVs into the circulation. Healthy individuals were subjected to an incremental exercise protocol of cycling or running until exhaustion, and EVs were isolated from blood plasma samples taken before, immediately after and 90 min after exercise. Small EVs with the size of 100-130 nm, that carried proteins characteristic of exosomes, were significantly increased immediately after cycling exercise and declined again within 90 min at rest. In response to treadmill running, elevation of small EVs was moderate but appeared more sustained. To delineate EV release kinetics, plasma samples were additionally taken at the end of each increment of the cycling exercise protocol. Release of small EVs into the circulation was initiated in an early phase of exercise, before the individual anaerobic threshold, which is marked by the rise of lactate. Taken together, our study revealed that exercise triggers a rapid release of EVs with the characteristic size of exosomes into the circulation, initiated in the aerobic phase of exercise. We hypothesize that EVs released during physical activity may participate in cell communication during exercise-mediated adaptation processes that involve signalling across tissues and organs.

Release of bulk cell free DNA during physical exercise occurs independent of extracellular vesicles.

PURPOSE: Strenuous exercise induces a rapid and transient elevation of cell free DNA (cfDNA) concentration in blood plasma. The detection of cfDNA in the presence of plasma nucleases could indicate an association of cfDNA with protective vesicular structures. Several cell types release extracellular vesicles (EVs), including exosomes and shedding microvesicles, which are known to mediate the exchange of proteins and nucleic acids (largely RNA) between cells. Here, we assessed whether EVs play a role in the exercise-dependent release of cfDNA in blood plasma. METHODS: Venous blood collected from healthy volunteers before and after incremental treadmill exercise was separated into vesicular (EV) and soluble fractions. Nuclear and mitochondrial DNA content in plasma supernatants and EV fractions was determined by quantitative real-time PCR (qPCR). RESULTS: We show that the majority of cfDNA is located in the plasma supernatants. Only minute amounts of DNA were observed in the EV-associated fractions including microvesicles and exosomes. Nuclear and mitochondrial DNA species differ in terms of their quantities in the several plasma fractions. CONCLUSIONS: Our results indicate that cfDNA liberated in response to acute physical exercise is not released by vesicular means and circulates in a soluble form in blood plasma which could indicate different biological functions exerted by cfDNA and EVs. The different nature of DNA species in plasma has major implications for the preparation of plasma and other bodily fluids prior to analysis.

Exercise-induced increases in cell free DNA in human plasma originate predominantly from cells of the haematopoietic lineage.

The role of cell free DNA (cfDNA) has been intensively discussed under various pathological conditions and after acute bouts of exercise. To date, there is still no conclusive evidence concerning the cellular origin of cfDNA and the entire mechanism leading to elevated cfDNA concentrations in human plasma and serum. Here, we investigated the cellular origin of cfDNA in sex-mismatched haematopoietic stem cell transplantation (HSCT) and liver transplantation (LT) patients by determining the relative proportion of Y-chromosomal to total nuclear cfDNA. Total nuclear cfDNA and Y-chromosomal cfDNA concentrations were determined in blood plasma before and after an incremental exercise test via quantitative real-time PCR (qPCR). Female HSCT patients showed high proportions of Y-chromosomal cfDNA. Both total nuclear and Y-chromosomal cfDNA increased significantly and in a highly correlated fashion due to exercise. In male HSCT patients with female donors less than 10% of the cfDNA was of Y-chromosomal origin at any point in time and even though the total amount of cfDNA increased during exercise, no increases in Y-chromosomal DNA could be detected. The percentage of Y-chromosomal cfDNA in female LT patients with male donors was very low and levels remained unchanged during exercise. This indicates that cells not derived from the bone marrow, in this case transplanted liver cells, represented only a minor fraction of cfDNA in blood plasma and were not released during acute physical exercise. Even though many physiological conditions may be altered in transplant patients versus healthy people, our results strongly suggest that cells from the haematopoietic lineage are the main source of cfDNA released during acute bouts of exercise.

Correlation between cell free DNA levels and medical evaluation of disease progression in systemic lupus erythematosus patients.

High levels of cell free DNA (cfDNA) in human blood plasma have been described in patients with autoimmune diseases. The aim of this study was to determine the levels of cfDNA in systemic lupus erythematosus (SLE) patients and to assess fluctuations of cfDNA concentrations compared to the course of disease progression under standard treatment. Therefore, nuclear cfDNA concentrations in plasma were measured in 59 SLE patients and 59 healthy controls. Follow-up blood plasma was collected from 27 of the 59 SLE patients. Patients were characterised by clinical parameters (antinuclear antibodies (ANA), anti-dsDNA-antibodies, C3, C4, and CRP), SLE disease activity index (SLEDAI) and medical therapy. Our results showed that cfDNA concentrations were significantly higher in SLE patients compared to healthy individuals. Levels of cfDNA assessed in serial samples correlated significantly with the medical evaluation of disease activity in SLE patients. Our results could implicate cfDNA as a global marker for disease activity.

Child-caregivers' body weight and habitual physical activity status is associated with overweight in kindergartners.

BACKGROUND: The aim of this study was to examine whether child-caregivers', both parents and kindergarten teachers, health parameters (age, weight status, habitual physical activity score) are significantly associated with the risk of overweight in young children. METHODS: We assessed the individual body mass index standard deviation score in a regional cross-sectional health study and matched a representative sample of 434 kindergartners aged 3 to 6-years with their caregivers' weight and habitual physical activity status. Furthermore, we identified factors associated with the general ability of child-caregivers to identify overweight in children, and the awareness to classify a child within the correct weight category. RESULTS: Our study confirmed most of the known associations between parental anthropometrics and psychosocial factors with childhood overweight and obesity. A significantly higher proportion of boys tended to be overweight or obese (p = 0.027) and parents were more likely to misclassified boys overweight as normal weight (OR: 1.86; 95% CI 1.21-2.86). Adjusted for confounders, logistic regression analysis revealed that kindergarten teachers' weight status (OR: 1.97; 95%-CI: 1.01-3.83) and habitual physical activity scores (OR: 2.32; 95%-CI: 1.10-4.92) were associated with children's weight status. CONCLUSIONS: Kindergarten teachers' weight and habitual physical activity score seem to be new independent risk factors for overweight in kindergartners 3 to 6-years of age. Our results suggest that the psychosocial, non-genetic association of non-parental child-caregivers on children's weight is relatively high and that the association of non-parental child-caregivers warrants further investigation.

Conventional and genetic talent identification in sports: will recent developments trace talent?

The purpose of talent identification (TI) is the earliest possible selection of auspicious athletes with the goal of systematically maximizing their potential. The literature proposes excellent reviews on various facets of talent research on different scientific issues such as sports sciences or genetics. However, the approaches of conventional and genetic testing have only been discussed separately by and for the respective groups of interest. In this article, we combine the discoveries of these disciplines into a single review to provide a comprehensive overview and elucidate the prevailing limitations. Fundamental problems in TI reside in the difficulties of defining the construct 'talent' or groups of different performance levels that represent the target variable of testing. Conventional and genetic testing reveal a number of methodological and technical limitations, and parallels are summarised in terms of the test designs, the point in time of testing, psychological skills or traits and unknown interactions between different variables. In conclusion, many deficiencies in the current talent research have gained attention. Alternative solutions include the talent development approach, while genetic testing is re-emphasised as a tool for risk stratification in sport participation. Future research needs to clearly define the group of interest and comprehensively implement all methodological improvement suggestions.

Direct measurement of cell-free DNA from serially collected capillary plasma during incremental exercise.

To investigate the kinetics of cell-free DNA (cfDNA) due to exercise, we established a direct real-time PCR for the quantification of cfDNA from unpurified capillary plasma by amplification of a 90- and a 222-bp multilocus L1PA2 sequence. Twenty-six male athletes performed an incremental treadmill test. For cfDNA measurement, capillary samples were collected serially from the fingertip preexercise, during, and several times postexercise. Venous blood was drawn before and immediately after exercise to compare capillary and venous cfDNA values. To elucidate the strongest association of cfDNA accumulations with either cardiorespiratory or metabolic function during exercise, capillary cfDNA values were correlated with standard measures like heart rate, oxygen consumption, or lactate concentrations. The venous cfDNA concentrations were significantly higher compared with the capillary plasma, but in both fractions cfDNA increased 9.8-fold and the values correlated significantly (r = 0.796). During incremental treadmill running, the capillary cfDNA concentrations increased nearly parallel to the lactate values. The values correlated best with heart rate and energy expenditure, followed by oxygen consumption, Borg values, and lactate levels (0.710 ≤ r ≥ 0.808). With this article, we present a sensitive procedure for the direct quantification of cfDNA in unpurified capillary plasma instead of purified venous plasma. Further studies should investigate the differences between capillary and venous cfDNA that might mirror different physiological mechanisms. Enhanced cardiorespiratory function during exercise might lead to the accumulation of cfDNA via the release of stress hormones that already increase at intensities below the anaerobic threshold. Furthermore, cfDNA might be released by neutrophil extracellular traps.

Direct quantification of cell-free, circulating DNA from unpurified plasma.

Cell-free DNA (cfDNA) in body tissues or fluids is extensively investigated in clinical medicine and other research fields. In this article we provide a direct quantitative real-time PCR (qPCR) as a sensitive tool for the measurement of cfDNA from plasma without previous DNA extraction, which is known to be accompanied by a reduction of DNA yield. The primer sets were designed to amplify a 90 and 222 bp multi-locus L1PA2 sequence. In the first module, cfDNA concentrations in unpurified plasma were compared to cfDNA concentrations in the eluate and the flow-through of the QIAamp DNA Blood Mini Kit and in the eluate of a phenol-chloroform isoamyl (PCI) based DNA extraction, to elucidate the DNA losses during extraction. The analyses revealed 2.79-fold higher cfDNA concentrations in unpurified plasma compared to the eluate of the QIAamp DNA Blood Mini Kit, while 36.7% of the total cfDNA were found in the flow-through. The PCI procedure only performed well on samples with high cfDNA concentrations, showing 87.4% of the concentrations measured in plasma. The DNA integrity strongly depended on the sample treatment. Further qualitative analyses indicated differing fractions of cfDNA fragment lengths in the eluate of both extraction methods. In the second module, cfDNA concentrations in the plasma of 74 coronary heart disease patients were compared to cfDNA concentrations of 74 healthy controls, using the direct L1PA2 qPCR for cfDNA quantification. The patient collective showed significantly higher cfDNA levels (mean (SD) 20.1 (23.8) ng/ml; range 5.1-183.0 ng/ml) compared to the healthy controls (9.7 (4.2) ng/ml; range 1.6-23.7 ng/ml). With our direct qPCR, we recommend a simple, economic and sensitive procedure for the quantification of cfDNA concentrations from plasma that might find broad applicability, if cfDNA became an established marker in the assessment of pathophysiological conditions.

Obesity prevalence and unfavorable health risk behaviors among German kindergarten teachers: cross-sectional results of the kindergarten teacher health study.

BACKGROUND: The aim of the study was to investigate obesity status and associated health risk behaviors in a sample of German kindergarten teachers. At present, such data are not available, despite the fact that kindergarten teachers educate children at a formative time in their lives. METHODS: Kindergarten teachers aged 18-62 years (n = 313) were invited to participate in the Kindergarten Teacher Health Study (KTHS) by completing a self-reported questionnaire. We analyzed their obesity status, health risk behaviors (i.e., habitual physical activity, screen time activities, eating behavior patterns, smoking), and their general ability to identify overweight children and the associated health risks of overweight and obesity based on special age- and sex-specific silhouettes. After adjusting for covariates, bivariate correlations were conducted for associations between body mass index (BMI) and health risk behaviors, while analyses of variance (ANOVAs) were used to analyze differences of health risk behaviors between BMI groups. Logistic regression analyses were conducted to predict determinants of kindergarten teachers who did not correctly identify the overweight silhouettes and their associated physical and mental health risks. Additionally, data regarding kindergarten teachers' weight status and smoking behavior were compared with nationally representative data from the 2009 Microcensus (n = 371310) using the Mann-Whitney U-test. RESULTS: The prevalence rates of overweight and obesity were 41.2% and 17.9%, respectively. The prevalence of obesity was significantly higher in kindergarten teachers (p < 0.001) compared to national Microcensus data. Only 44.6% of teachers were able to identify overweight children correctly. The fact that being overweight is associated with physical and mental health risks was only reported by 40.1% and 21.2% of teachers, respectively. Older kindergarten teachers were more likely to misclassify the overweight silhouettes, while younger, normal-weight, and overweight kindergarten teachers were more likely to underestimate the associated health risks. Obese kindergarten teachers reported spending more time in front of computer and television screens than their normal-weight counterparts, especially on weekends. In addition, obese kindergarten teachers reported eating less often with their families and more frequently reported watching television during meals. CONCLUSIONS: Advanced monitoring and multifaceted interventions to improve the health behaviors of kindergarten teachers should be given high priority. Because kindergarten teachers' behavioral modeling presumably mediates children's health behaviors, additional research is needed about kindergarten teachers' health and its proposed interaction with children's health.

Psychodynamic Motivation and Training program (PMT) for the secondary prevention in patients with stable coronary heart disease: study protocol for a randomized controlled trial of feasibility and effects.

BACKGROUND: Nonpharmacological secondary prevention of coronary heart disease is considered a safe and effective measure to substantially reduce mortality. Despite the effectiveness of lifestyle changes, the compliance rate of patients is very low mainly due to psychosocial barriers. Psychotherapeutic approaches that address how persons think about themselves and their behaviors appear to have a significant potential for improving health behavior. METHODS/DESIGN: Against this background, our study aims to examine the feasibility and effects of a Psychodynamic Motivation and Training program (PMT) as compared to one session of advice in exercise training (EX) and treatment as usual (TAU). For that purpose, 90 patients with stable coronary heart disease and a physically inactive lifestyle will be randomly assigned to the three groups (each with n = 30). The primary outcome is the change in the individual anaerobic threshold as determined by spiroergometry from baseline to six month follow-up. Secondary endpoints include change in endothelial function, biomarkers of inflammation and oxidative stress, quality of life, symptoms of fatigue, illness perception and feasibility of the treatment approach. We hypothesize that physical fitness will improve more in PMT than in EX and TAU, with PMT and EX more than TAU, and that the effects will be more pronounced for participants with current mental or psychosocial distress. DISCUSSION: The results of the study will help to determine the effectiveness of a psychodynamic lifestyle change approach and to identify measures for designing specifically tailored interventions to improve compliance with cardiovascular prevention. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01445808.

Circulating cell-free DNA: an up-coming molecular marker in exercise physiology.

The phenomenon of circulating cell-free DNA (cfDNA) concentrations is of importance for many biomedical disciplines including the field of exercise physiology. Increases of cfDNA due to exercise are described to be a potential hallmark for the overtraining syndrome and might be related to, or trigger adaptations of, immune function induced by strenuous exercise. At the same time, exercise provides a practicable model for studying the phenomenon of cfDNA that is described to be of pathophysiological relevance for different topics in clinical medicine like autoimmune diseases and cancer. In this review, we are summarizing the current knowledge of exercise-based acute and chronic alterations in cfDNA levels and their physiological significance. The effects of acute exercise on cfDNA concentrations have been investigated in resistance exercises and in continuous, stepwise and interval endurance exercises of different durations. cfDNA concentrations peaked immediately after acute exercise and showed a rapid return to baseline levels. Typical markers of skeletal muscle damage (creatine kinase, uric acid, C-reactive protein) show delayed kinetics compared with the cfDNA peak response. Exercise parameters such as intensity, duration or average energy expenditure do not explain the extent of increasing cfDNA concentrations after strenuous exercise. This could be due to complex processes inside the human organism during and after physical activity. Therefore, we hypothesize composite effects of different physiological stress parameters that come along with exercise to be responsible for increasing cfDNA concentrations. We suggest that due to acute stress, cfDNA levels increase rapidly by a spontaneous active or passive release mechanism that is not yet known. As a result of the rapid and parallel increase of cfDNA and lactate in an incremental treadmill test leading to exhaustion within 15-20 minutes, it is unlikely that cfDNA is released into the plasma by typical necrosis or apoptosis of cells in acute exercise settings. Recently, rapid DNA release mechanisms of activated immune-competent cells like NETosis (pathogen-induced cell death including the release of neutrophil extracellular traps [NETs]) have been discovered. cfDNA accumulations might comprise a similar kind of cell death including trap formation or an active release of cfDNA. Just like chronic diseases, chronic high-intensity resistance training protocols induced persistent increases of cfDNA levels. Chronic, strenuous exercise protocols, either long-duration endurance exercise or regular high-intensity workouts, induce chronic inflammation that might lead to a slow, constant release of DNA. This could be due to mechanisms of cell death like apoptosis or necrosis. Yet, it has neither been implicated nor proven sufficiently whether cfDNA can serve as a marker for overtraining. The relevance of cfDNA with regard to overtraining status, performance level, and the degree of physical exhaustion still remains unclear. Longitudinal studies are required that take into account standardized and controlled exercise, serial blood sampling, and large and homogeneous cohorts of different athletic achievement. Furthermore, it is important to establish standardized laboratory procedures for the measurement of genomic cfDNA concentrations by quantitative real-time polymerase chain reaction (PCR). We introduce a new hypothesis based on acute exercise and chronic exposure to stress, and rapid active and passive chronic release of cfDNA fragments into the circulation.

Role of reactive oxygen species in the regulation of HIF-1 by prolyl hydroxylase 2 under mild hypoxia.

The function and survival of eukaryotic cells depends on a constant and sufficient oxygen supply. Cells recognize and respond to hypoxia by accumulation of the transcription factor hypoxia-inducible factor 1 (HIF-1), composed of an oxygen-sensitive HIF-1α and a constitutive HIF-1ß subunit. Besides physiology, HIF-1 induction is involved in major pathological processes such as cardiovascular disease, inflammation and cancer, which are associated with the formation of reactive oxygen species (ROS). ROS have been reported to affect HIF-1 activity but the role for ROS in regulating HIF-1 has not been definitely settled. In order to shed light on the redox-regulation of HIF-1 by ROS, we studied the impact of exogenous ROS treatment (H(2)O(2)) on HIF-1α and HIF-1 regulatory protein prolyl hydroxylase 2 (PHD2) in the human osteosarcoma cell line U2OS. At early reaction periods, H(2)O(2) induced HIF-1α but at prolonged observation phases the opposite occurred. Herein, modulation of PHD activity appeared to be the key element, because knockdown and inhibition of the PHD2 prevented reduction of HIF-1α. However, H(2)O(2) treatment constantly suppressed HIF-1 transactivation at all time-points. Our data indicate a dual redox regulation of HIF-1α protein amount with a constant suppression of HIF-1 target gene expression by ROS.

Role of N-acetyl-N-nitroso-tryptophan as nitric oxide donor in the modulation of HIF-1-dependent signaling.

N-Acetyl-N-nitroso-tryptophan (NANT) is well known for its capacity to generate nitric oxide (NO)-releasing compounds. It is unknown, however, whether NANT can be successfully applied as a precursor of NO in a complex biological environment such as a cell culture system. NO donors can be useful to induce the transcription factor hypoxia-inducible factor 1 (HIF-1) that coordinates the protection of cells and tissues from the lack of oxygen, termed hypoxia. HIF-1 degradation is controlled by prolyl hydroxylase 2 (PHD2) which needs to be inhibited for HIF-1 accumulation. Here, the effects of NANT in inhibiting recombinant PHD2 and up-regulating of HIF-1 and HIF-1-mediated carboanhydrase-9 (CA9) mRNA expression were compared in living cells with the NO donors N-nitrosomelatonin (NOMela) and S-nitrosoglutathione (GSNO) under normoxic and hypoxic conditions. In contrast to GSNO, NANT was similar to NOMela being highly effective in inhibiting recombinant PHD2. NANT-mediated activation of HIF-1 in oxygenated cells was comparable to hypoxic activation of HIF-1 in all cases. In contrast, under hypoxia NANT was able to boost hypoxic cellular HIF-1 levels by further reducing the activity of cellular PHD2. The strong increase of HIF-dependent CA9 mRNA expression demonstrated that NANT-induced HIF-1 was transcriptionally active. Finally, the efficacy of NANT to increase both HIF-1 and CA9 mRNA did not depend on the absolute conformation of the tryptophan moiety. In conclusion, NANT appears to be an excellent NO donor for cells in culture and l-NANT should be useful for in vivo animal studies.

Oxygen-sensing under the influence of nitric oxide.

The transcription factor complex Hypoxia inducible factor 1 (HIF-1) controls the expression of most genes involved in adaptation to hypoxic conditions. Oxygen-dependency is maintained by prolyl- and asparagyl-4-hydroxylases (PHDs/FIH-1) belonging to the superfamily of iron(II) and 2-oxoglutarate dependent dioxygenases. Hydroxylation of the HIF-1alpha subunit by PHDs and FIH-1 leads to its degradation and inactivation. By hydroxylating HIF-1alpha in an oxygen-dependent manner PHDs and FIH-1 function as oxygen-sensing enzymes of HIF signalling. Besides molecular oxygen nitric oxide (NO), a mediator of the inflammatory response, can regulate HIF-1alpha accumulation, HIF-1 activity and HIF-1 dependent target gene expression. Recent studies addressing regulation of HIF-1 by NO revealed a complex and paradoxical picture. Acute exposure of cells to high doses of NO increased HIF-1alpha levels irrespective of the residing oxygen concentration whereas prolonged exposure to NO or low doses of this radical reduced HIF-1alpha accumulation even under hypoxic conditions. Several mechanisms were found to contribute to this paradoxical role of NO in regulating HIF-1. More recent studies support the view that NO regulates HIF-1 by modulating the activity of the oxygen-sensor enzymes PHDs and FIH-1. NO dependent HIF-1alpha accumulation under normoxia was due to direct inhibition of PHDs and FIH-1 most likely by competitive binding of NO to the ferrous iron in the catalytically active center of the enzymes. In contrast, reduced HIF-1alpha accumulation by NO under hypoxia was mainly due to enhanced HIF-1alpha degradation by induction of PHD activity. Three major mechanisms are discussed to be involved in enhancing the PHD activity despite the lack of oxygen: (1) NO mediated induction of a HIF-1 dependent feedback loop leading to newly expressed PHD2 and enhanced nuclear localization, (2) O2-redistribution towards PHDs after inhibition of mitochondrial respiration by NO, (3) reactivation of PHD activity by a NO mediated increase of iron and 2-oxoglutarate and/or involvement of reactive oxygen and/or nitrogen species.